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Based on the open-source computational fluid dynamics (CFD) platform, OpenFOAM, two numerical simulation methods for gusty inflow characterization and gust response prediction are implemented by solving the fundamental incompressible unsteady Reynolds averaged Navier–Stokes (URANS) equations. One is the Field Velocity Method (FVM) and the other is the Oscillating Vane Method (OVM). The gust velocity field is characterized and the aerodynamic responses of some airfoils under the Sears-type sinusoidal gusts are predicted by both gust simulation methods. The results indicate that both methods are capable of obtaining satisfactory gusty inflow conditions as expected as well as the airfoil aerodynamic responses. Comparatively, from the perspective of computing cost, the FVM is more advantageous in reducing the computational resources than the OVM while simultaneously ensuring the computational accuracy.

Mycobacterium tuberculosis Rv3717 has been identified as a zinc-dependent amidase which can hydrolyze peptidoglycan (PG). To demonstrate the relationship of Rv3717 and cell division, in this study, Rv3717 gene was first amplified and expressed and the resulting protein was purified by using a His-tagged approach. M. smegmatis mc2155, a fast-growing and nonpathogenic mycobacterium was used to evaluate the effect of Rv3717 on cell division. Scan electron microscope (SEM) results indicated that M. smegmatis with division site was more exhibited and some of the cells turned larger in size after Rv3717 treatment. Transmission electron microscope (TEM) results revealed that MSMEG_6281 gene knockout strain named M sm-ΔM_6281 (MSMEG_6281 in M. smegmatis mc2155 is the homologous gene of Rv3717) tended to have a division defect with a severely abnormal morphology, and division septa were distorted. Gene expression analysis indicated also that the gene involved in cell division such as M. smegmatis ftsZ was significantly up-regulated with treatment time. The findings demonstrated that physiological role of Rv3717 was related to cell division and regulated possibly division septum formation. Further, fibronectin (Fn) binding ability of Rv3717 was evaluated by protein binding experiment, and the results confirmed the interaction of Rv3717 with Fn in a dose dependent manner. We found also that the invasion rate of M. sm-ΔM_6281 to A549 cells was reduced by 59% compared to the control strain, and the invasion defect could be rescued by Rv3717 addition. RT-PCR results showed that M. smegmatis fbpC were up-regulated after Rv3717 addition. These clues may be significant to explore roles of Rv3717 in growth and colonization of mycobacteria.

Mycobacterium smegmatis MSMEG_6281, a peptidoglycan (PG) amidase, is essential in maintaining cell wall integrity. To address the potential roles during the MSMEG_6281-mediated biological process, we compared proteomes from wild-type M.smegmatis and MSMEG_6281 gene knockout strain (M.sm-ΔM_6281) using LC-MS/MS analysis. Peptide analysis revealed that 851 proteins were differentially produced with at least 1.2-fold changes, including some proteins involved in fatty acid metabolism such as acyl-CoA synthase, acyl-CoA dehydrogenase, MCE-family proteins, ATP-binding cassette (ABC) transporters, and MmpL4. Some proteins related to fatty acid degradation were enriched through protein-protein interaction analysis. Therefore, proteomic data showed that a lack of MSMEG_6281 affected fatty acid metabolism. Mycobacteria can produce diverse lipid molecules ranging from single fatty acids to highly complex mycolic acids, and mycobacterial surface-exposed lipids may impact biofilm formation. In this study, we also assessed the effects of MSMEG_6281 on biofilm phenotype using semi-quantitative and morphology analysis methods. These results found that M.sm-ΔM_6281 exhibited a delayed biofilm phenotype compared to that of the wild-type M.smegmatis, and the changes were recovered when PG amidase was rescued in a ΔM_6281::Rv3717 strain. Our results demonstrated that MSMEG_6281 impacts fatty acid metabolism and further interferes with biofilm formation. These results provide a clue to study the effects of PG amidase on mycobacterial pathogenicity.

Mycolyl-arabinogalactan-peptidoglycan (mAGP) is the major content of the mycobacterium cell wall structure and essential for mycobacterial survival. Peptidoglycan (PG) plays an important role in maintenance of cell division, cell wall integrity and pathogenesis. Mycobacterium smegmatis MSMEG_6281, a peptidoglycan amidase, is vital for mycobacterial cell division. However, the effects of MSMEG_6281on cell wall integrity and mycobacterial virulence remain unknown. In the current study, we demonstrate that MSMEG_6281gene knockout in M.smegmatis alters the microbiological characteristics. Our results revealed that MSMEG_6281gene knockout bacteria (M. sm-ΔM_6281) lost their acid-fastness, increased their sensitivity to lipophilic compounds and presented an abnormal morphology. Our results revealed that MSMEG_6281was related to maintaining the cell wall integrity. Furthermore, we investigated the effects of MSMEG_6281 inactivation on mycobacterial virulence using mice models infected by different M.smegmatis strains. MSMEG_6281 inactivation in the M sm-ΔM_6281 infected group caused less mycobacterial colonization, reduced pathological signs, decreased the anti-microbial enzymes production including iNOS and β-defensins in mouse lungs. Moreover, IL-1β and TLR2 expression were significantly down-regulated, while the production of IFN-γ and TNF-α was up-regulated. These findings indicated the diversity of host immune responses induced by different strains of M.smegmatis, suggesting that MSMEG_6281 inactivation impact mycobacterial virulence. In conclusion, the MSMEG_6281 protein plays important roles in maintaining cell wall integrity and mycobacterial virulence.